Parides ascanius (Lepidoptera: Papilionidae) is a butterfly endemic to the sand forests (“restingas”) of one of the most populated areas of Brazil (from Rio de Janeiro state to South Espírito Santo state), and was the first invertebrate officially recognized as being threatened in Brazil. Here we present a panel of eight polymorphic microsatellite loci and partial sequences of mitochondrial gene COI aiming to characterize this butterfly’s genetic diversity and understand its distribution among the extant populations. We estimate FST metrics, migration rates, cluster assignment, and spatial structure of genetic diversity. FST and statistics indicate low genetic structure and no evidence for endogamy, with all populations connected by high migration rates. Seven populations have low permanence rates (68–75 %) with increased migration probabilities for all populations. One population displays higher permanence rate (87.7 %), as the metropolitan matrix isolates it. Spatial analysis shows a global structure around the city of Rio de Janeiro and the Guanabara Bay; assignment analysis recovers six clusters evenly spread among sampled populations. These findings are consistent with a natural scenario of metapopulation dynamics for P. ascanius, with low genetic diversity and no endogamy, but progressively isolated by the metropolitan matrix. Conservation efforts should focus in connecting the isolated population, broaden the searches for new populations, and preserve all extant habitat patches where P. ascanius still occurs. © 2015, Springer Science+Business Media Dordrecht.173533546